Since epidemiological studies indicate that more than 80% of infections with HIV are acquired at mucosal surfaces, a safe and effective vaccine must have the properties to stimulate both the mucosal and systemic immune responses. Infection with poliovirus results in the stimulation of both humoral and cell mediated components of the systemic immune systems. During the last funding period, we developed recombinant poliovirus genomes, termed replicons, in which the capsid gene has been substituted with HIV/SIV genes. A complementation system to provide capsid proteins in trans has been developed to encapsidate replicons. Immunization of mice or primates with encapsidated replicons results in an immune response against the target HIV/SIV protein. The goal of the proposed experiments is to further optimize the expression of HIV proteins, and enhance the immunogenicity of the replicons. The Specific Aims are: Specific Aim 1: To optimize the expression of HIV envelope from poliovirus replicons. To do this, we will modify the HIV Env gene to encode different secretory translocation (signal sequences). Modifications of the replicon vector will be made to allow increased expression of HIV Env. Specific Aim 2: To enhance infectivity of encapsidated replicons. To enhance interactions with the target cell, replicons will be tested that contain the B subunit of cholera toxin (CTB) conjugated to the capsid, or the capsid modified to express a cell surface integrin binding motif (RGD). Based on previous studies, we will confirm that replicons formulated into liposome-cochleates can circumvent receptor mediated infection. We will determine if the modified replicons show enhanced infection and expression of foreign proteins in primary cultures of macrophages of dendritic cells. Specific Aim 3: To test replicon formulations for the ability to induce immune responses. The modified replicons described in Aims 1 and 2 will be tested for immunogenicity in transgenic mice that contain the receptor poliovirus. Replicons encoding cytokines will be evaluated for enhancement of the immunogenicity. The optimized formulation will be evaluated in primates with Drs. Fultz (Project 3) and Letvin (Project 4). Studies conducted during the last funding period have established the feasibility of using replicons as vaccine vectors. The experiments designed to optimize the expression of HIV/SIV proteins and enhance immunogenicity are essential for the complete evaluation of vaccine strategies required for future clinical trials.